The membrane machinery by which beta-adrenergic catecholamines and many other hormones regulate cyclic AMP synthesis in animal cells is composed of three separable components: Hormone receptors (R), catalytic adenylate cyclase (C) and a component required for functional coupling of R and C, termed N because it mediates regulation of adenylate cyclase by guanyl nucleotides. We propose to define the structure of N, purify it, and probe its interactions with R and C, using the following tools: 1. Cholera toxin, which catalyzes ADP-ribosylation of a 42,000 molecular weight peptide subunit of N. Because the toxin substrate can be specifically radiolabelled with P32-NAD ion, it is the only macromolecular component of hormone-sensitive adenylate cyclase that can be detected and characterized by techniques that may destroy enzymatic or ligand binding activities. Such techniques, to be used in this project, include proteolytic digestion, covalent chemical modification, polyacrylamide gel electrophoresis under denaturing conditions and chemical cross-linking with bifunctional agents. 2. Guanyl nucleotide affinity columns, which specifically bind N and facilitate its purification. 3. Cells specifically deficient in one or more components of hormone-sensitive adenylate cyclase, including S49 lymphoma variants deficient in N or beta-adrenergic R and human erythrocytes, which possess N but lack C and beta-adrenergic R. These cells allow in vitro complementation studies of interactions between N and other components, and also provide sensitive functional assays for N. Using these complementary approaches we will elucidate the structure and function of a membrane molecule that is essential for transduction of hormonal signals across the plasma membrane.